CRISPR-Cas9-mediated Large Cluster Deletion and Multiplex Genome Editing in Paenibacillus polymyxa

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) system has rapidly advanced genetic engineering research. The system has been applied for different genetic engineering purposes in multiple organisms including the quite rarely explored Paenibacillus polymyxa. Only limited studies on CRISPR-based system have been described for this highly interesting and versatile bacterium. Here, we demonstrated the utilization of a Cas9-based system to realize 32.8 kb deletion of genomic region by using a single targeting sgRNA. Large cluster deletion was successfully performed with remarkable efficiency of 97 %. Furthermore, we also exploited the system for multiplexing by editing of two distantly located genes at once. We investigated double gene knockouts as well as simultaneous gene integrations and reached editing efficiencies of 78 % and 50 %, respectively. The findings reported in this study are anticipated to accelerate future research in P. polymyxa and related species.

Insights in the complex DegU, DegS, Spo0A regulation system of Paenibacillus polymyxa by CRISPR-Cas9-based targeted point mutations

“…In previous studies, we have developed efficient CRISPR-Cas-based systems to facilitate gene deletions and regulations in P. polymyxa DSM 365. 15,18,41,42 Here, we demonstrated the reliability of the system to introduce targeted point mutations into the DegU, DegS, and Spo0A regulatory proteins, which are rarely investigated in P. polymyxa . Using the defined mutants generated by targeted integration of specific single nucleotide polymorphisms (SNPs) via CRISPR-Cas9, we propose the ability of DegU, DegS, and Spo0A in P. polymyxa to act as a phosphorelay-dependent multi-stage switch, which gradually controls various adaptive traits (Figure 6).…”